Peter J. Steenbergen

Acute Activation of Hippocampal Glucocorticoid Receptors Results in Different Waves of Gene Expression Throughout Time

Several aspects of hippocampal cell function are influenced by adrenal‐secreted glucocorticoids in a delayed, genomic fashion. Previously, we used Serial Analysis of Gene Expression to identify glucocorticoid receptor (GR)‐induced transcriptional changes in the hippocampus at a fixed time point. However, because changes in mRNA levels are transient and most likely precede the effects on hippocampal cell function, the aim of the current study was to assess the transcriptional changes in a broader time window by generating a time curve of GR‐mediated gene expression changes. Therefore, we used rat hippocampal slices obtained from adrenalectomised rats, substituted in vivo with low corticosterone pellets, predominantly occupying the hippocampal mineralocorticoid receptors. To activate GR, slices were treated in vitro with a high (100 nM) dose of corticosterone and gene expression was profiled 1, 3 and 5 h after GR‐activation. Using Affymetrix GeneChips, a striking pattern with different waves of gene expression was observed, shifting from exclusively down‐regulated genes 1 h after GR‐activation to both up and down regulated genes 3 h after GR‐activation. After 5 h, the response was almost back to baseline. Additionally, real‐time quantitative polymerase chain reaction was used for validation of a selection of responsive genes including genes involved in neurotransmission and synaptic plasticity such as the corticotropin releasing hormone receptor 1, monoamine oxidase A, LIMK1 and calmodulin 2. This permitted confirmation of GR‐responsiveness of 15 out of 18 selected genes. In conclusion, direct activation of GR in hippocampal slices results in transient changes in gene expression. The pattern in which gene expression was modulated suggests that the fast genomic effects of glucocorticoids may be realised via transrepression, preceding a later wave of transactivation. Furthermore, we identified a number of interesting candidate genes which may underlie the glucocorticoid‐mediated effects on hippocampal cell function.

The use of the zebrafish model in stress research.

The study of the causes and mechanisms underlying psychiatric disorders requires the use of non-human models for the test of scientific hypotheses as well as for use in pre-clinical drug screening and discovery. This review argues in favor of the use of zebrafish as a novel animal model to study the impact of early (stressful) experiences on the development of differential stress phenotypes in later life. This phenomenon is evolutionary conserved among several vertebrate species and has relevance to the etiology of psychiatric disorders. Why do we need novel animal models? Although significant progress has been achieved with the use of traditional mammalian models, there are major pitfalls associated with their use that impedes progress on two major fronts: 1) uncovering of the molecular mechanisms underlying aspects of compromised (stress-exposed) brain development relevant to the etiology of psychiatric disorders, and 2) ability to develop high-throughput technology for drug discovery in the field of psychiatry. The zebrafish model helps resolve these issues. Here we present a conceptual framework for the use of zebrafish in stress research and psychiatry by addressing three specific domains of application: 1) stress research, 2) human disease mechanisms, and 3) drug discovery. We also present novel methodologies associated with the development of the zebrafish stress model and discuss how such methodologies can contribute to remove the main bottleneck in the field of drug discovery.

Patterns of avoidance behaviours in the light/dark preference test in young juvenile zebrafish: A pharmacological study

The light/dark preference test is commonly used to assess anxiety-like phenotypes and validate the pharmacological effects of neuroactive compounds. This test has been recently adapted for adult zebrafish but has not yet been characterized and pharmacologically validated for young juvenile zebrafish. In the present study, we provide a detailed description of the pattern of exploratory behaviours encountered in juvenile zebrafish when exposed to the light/dark preference test. We report that juveniles display strong dark-avoidance behaviours in this test. Specifically, juveniles spent significantly less time, displayed high latency to enter and moved significantly less in the dark compartment relative to the white compartment of the testing apparatus. The expression of these dark-avoidance behaviours was significantly attenuated and increased by commonly used anxiolytic (diazepam, buspirone, ethanol) and anxiogenic (caffeine but not FG-7142) drugs, respectively. We also show that the expression of dark-avoidance behaviours can be significantly reduced in a manner similar to what is achieved with anxiolytic drugs, simply by decreasing the contrast between the white and dark zones, which made the dark zone less dark. Taken together, these findings provide the first pharmacological validation of the light/dark preference test for juvenile zebrafish and ascertain the nature of dark-avoidance behaviours as anxiety-like behaviours in young juvenile zebrafish. This behavioural-based assay is also versatile and can accommodate drug screening of both anxiolytic and anxiogenic compounds while eventually amenable to automation and high-throughput capacity in a near future.

Expression profiling in laser-microdissected hippocampal subregions in rat brain reveals large subregion-specific differences in expression.

Expression profiling in the hippocampus is hampered by its cellular heterogeneity. The aim of this study was to evaluate the feasibility of using laser‐microdissected hippocampal subregions for expression profiling to improve detection of transcripts with a subregion‐specific expression. Cornu ammonis (CA)3 and dentate gyrus (DG) subregions were isolated from rat brain slices using laser microdissection, subjected to two rounds of linear amplification and hybridized to rat GeneChips containing approximately 8000 transcripts (RG_U34A; Affymetrix). Analysis of the data using significance analysis of microarrays revealed 724 genes with a significant difference in expression between CA3 and DG with a false discovery rate of 2.1%, of which 264 had higher expression in DG and 460 in CA3. Several transcripts with known differential expression between the subregions were included in the dataset, as well as numerous novel mRNAs and expressed sequence tags. Sorting of the differentially expressed genes according to gene ontology classification revealed that genes involved in glycolysis and general metabolism, neurogenesis and cell adhesion were consistently expressed at higher levels in CA3. Conversely, a large cluster of genes involved in protein biosynthesis were expressed at higher levels in DG. In situ hybridization was used to validate differential expression of a selection of genes. The results of this study demonstrate that the combination of laser microdissection and GeneChip technology is both technically feasible and very promising. Besides providing an extensive inventory of genes showing differential expression between CA3 and DG, this study supports the idea that profiling in hippocampal subregions should improve detection of genes with a subregion‐specific expression or regulation.

Incorporation of an invasive plant into a native insect herbivore food web

The integration of invasive species into native food webs represent multifarious dynamics of ecological and evolutionary processes. We document incorporation of Prunus serotina (black cherry) into native insect food webs. We find that P. serotina harbours a herbivore community less dense but more diverse than its native relative, P. padus (bird cherry), with similar proportions of specialists and generalists. While herbivory on P. padus remained stable over the past century, that on P. serotina gradually doubled. We show that P. serotina may have evolved changes in investment in cyanogenic glycosides compared with its native range. In the leaf beetle Gonioctena quinquepunctata, recently shifted from native Sorbus aucuparia to P. serotina, we find divergent host preferences on Sorbus- versus Prunus-derived populations, and weak host-specific differentiation among 380 individuals genotyped for 119 SNP loci. We conclude that evolutionary processes may generate a specialized herbivore community on an invasive plant, allowing prognoses of reduced invasiveness over time. On the basis of the results presented here, we would like to caution that manual control might have the adverse effect of a slowing down of processes of adaptation, and a delay in the decline of the invasive character of P. serotina.

Long‐lasting effects of dexamethasone on immune cells and wound healing in the zebrafish

This study assessed the lasting impact of dexamethasone (DEX) exposure during early development on tissue repair capacity at later life stages (5, 14, and 24 days post fertilization [dpf]) in zebrafish larvae. Using the caudal fin amputation model, we show that prior exposure to DEX significantly delays but does not prevent wound healing at all life stages studied. DEX‐induced impairments on wound healing were fully restored to normal levels with longer post amputation recovery time. Further analyses revealed that DEX mainly exerted its detrimental effects in the early phase (0–5 hours) of wound‐healing process. Specifically, we observed the following events: (1) massive amount of cell death both by necrosis and apoptosis; (2) significant reduction in the number as well as misplacement of macrophages at the wound site; (3) aberrant migration and misplacement of neutrophils and macrophages at the wound site. These events were accompanied by significant (likely compensatory) changes in the expression of genes involved in tissue patterning, including up‐regulation of FKBP5 6 hours post DEX exposure and that of Wnt3a and RARγ at 24 hours post amputation. Taken together, this study provides evidence that DEX exposure during early sensitive periods of development appears to cause permanent alterations in the cellular/molecular immune processes that are involved in the early phase of wound healing in zebrafish. These findings are consistent with previous studies showing that antenatal course of DEX is associated with immediate and lasting alterations of the immune system in rodent models and humans. Therefore, the current findings support the use of the larval zebrafish model to study the impact of stress and stress hormone exposure in immature organisms on health risks in later life.

Acute Exposure to Dexamethasone in Early‐Life has Enduring Effects on Immune Cell Migration and Delayed Wound Healing Long after Treatment in Zebrafish

This study assessed the lasting impact of dexamethasone (DEX) exposure during early development on tissue repair capacity at later life stages (5, 14, and 24 days post fertilization [dpf]) in zebrafish larvae. Using the caudal fin amputation model, we show that prior exposure to DEX significantly delays but does not prevent wound healing at all life stages studied. DEX‐induced impairments on wound healing were fully restored to normal levels with longer post amputation recovery time. Further analyses revealed that DEX mainly exerted its detrimental effects in the early phase (0–5 hours) of wound‐healing process. Specifically, we observed the following events: (1) massive amount of cell death both by necrosis and apoptosis; (2) significant reduction in the number as well as misplacement of macrophages at the wound site; (3) aberrant migration and misplacement of neutrophils and macrophages at the wound site. These events were accompanied by significant (likely compensatory) changes in the expression of genes involved in tissue patterning, including up‐regulation of FKBP5 6 hours post DEX exposure and that of Wnt3a and RARγ at 24 hours post amputation. Taken together, this study provides evidence that DEX exposure during early sensitive periods of development appears to cause permanent alterations in the cellular/molecular immune processes that are involved in the early phase of wound healing in zebrafish. These findings are consistent with previous studies showing that antenatal course of DEX is associated with immediate and lasting alterations of the immune system in rodent models and humans. Therefore, the current findings support the use of the larval zebrafish model to study the impact of stress and stress hormone exposure in immature organisms on health risks in later life.

Response to: Responses of larval zebrafish to low pH immersion assay. Comment on Lopez-Luna et al.

[Lopez-Luna et al. (2017a)][1] investigated the utility of using larval zebrafish as a replacement for adults in nociceptive testing. Five days post-fertilisation larvae were held in a 25-well plate and monitored using a video tracking system. Either larvae were undisturbed or system water was added

Response of zebrafish larvae to mild electrical stimuli: A 96-well setup for behavioural screening

BACKGROUND Zebrafish larvae have a high potential as model system to replace rodents, especially in screening and drug discovery applications. However, an experimental setup to deliver mild electrical stimuli with simultaneous high throughput behavioural tracking has not yet been described. NEW METHOD A new tool was designed, making the delivery of electrical stimuli in a 96-well plate format possible. Using custom made electrode clips that can be slid over the walls of a square 96-well plate, 80 larvae could be tested simultaneously and behavioural responses recorded. RESULTS As proof of principle, two applications were tested: 1) The behavioural response after a single stimulus and the effect of buprenorphine on this response. 2) Habituation of locomotor activity to multiple stimuli and the involvement of the NMDA receptor. Reduced locomotor activity was observed after a single 5 V stimulus, however not with lower intensity stimuli. Pre-treatment with the analgesic buprenorphine prevented this response. Specificity of buprenorphine was confirmed using the antagonist naloxone. Habituation of locomotor activity was seen in response to multiple stimuli, depending on the inter stimulus interval. Treatment with the NMDA receptor antagonist memantine disrupted behavioural habituation. COMPARISON WITH EXISTING METHODS The equipment and setup described here are the first of its kind using a 96-well plate format, thereby increasing the potential throughput in screening applications using zebrafish larvae. CONCLUSION The combination of the described electrode clips for stimulus delivery and behavioural tracking allows for the use of zebrafish larvae in a new array of medium to high throughput applications.